|Product Includes||Quantity (with Count)||Storage Temp|
|Reduced Glutathione Standard||1 x 1 ea||-20°C|
|Glutathione-S-Transferase||1 x 1 ea||-20°C|
|Tris Assay Buffer||1 x 25 ml||+4°C|
|Digitonin Lysis Buffer||1 x 11 ml||-20°C|
|Monochlorobimane||1 x 1 ea||-20°C|
Upon receipt, Tris Assay Buffer (#13865) should be removed from #13859 and stored at 4°C. And remaining components should be stored at -20ºC.
Reduced Glutathione Working Solution as follows (see Table 1).
Table 1: GSH Working Solution for one 96-well plate (100 assays)
|Kit Component||Volume (µl)|
|Tris Assay Buffer||4930|
NOTE: To create a GSH standard curve, prepare a 100 µM GSH Standard Solution by diluting the 100 mM Reduced Glutathione Standard 1:1000 in Tris Assay Buffer. Run the standard curve in triplicate using 50 µl of standard per well. Create a five point standard curve by starting at 100 µM and using a 1:2 serial dilution in Tris Assay Buffer. Include a blank control. The GSH Standard Solution is not stable at this concentration and should be used within 2 hours or discarded.
Additional Reagents (not supplied): DMSO ( #12611)
NOTE: If using suspended or detached cells, collect and centrifuge the cell suspension for 5 min at 1,200 rpm. Discard the supernatant and add Digitonin Lysis Buffer to the cell pellet.
NOTE: use a 10 kDa MWCO centrifugal filter and follow the manufacturer’s instructions for cells that require an additional filtration step.
Table 2: Detection and Standard Curve Assays
|Sample||Test Sample (µl)||GSH Standard (µl)||Working Solution (µl)||Assay Buffer (µl)||Total Volume (µl)|
|Sample||1-50||-||50||50 minus sample volume||100|
The following procedure measures reduced glutathione in live cells using a 96-well plate. The same protocol can be modified for fluorescent imaging and flow cytometry detection.
NOTE: For best results, a cell number titration is recommended to determine the optimal cell seeding density. We recommend not exceeding 1x105 cells per well.
posted June 2020
Protocol Id: 2050
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